Force generation by kinesin and myosin cytoskeletal motor ... · 28/2/2013 · A Kinesin-5 Eg5 B...
Transcript of Force generation by kinesin and myosin cytoskeletal motor ... · 28/2/2013 · A Kinesin-5 Eg5 B...
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Force generation by kinesin and myosin cytoskeletalmotor proteins
F. Jon Kull1 and Sharyn A. Endow2,*1Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA2Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
*Author for correspondence ([email protected])
Journal of Cell Science 126, 1–11� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.103911
SummaryKinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. Howdo motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles andconformational changes observed in crystal structures. Distortion or twisting of the central b-sheet – proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins – is shown in a movie depicting the
transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governsATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor powerstrokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing.
Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesinsby induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specificallyincreases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further
study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treathuman disease.
Key words: Motor proteins, Kinesins, Myosins, Force generation, Mechanochemical cycles, Kinesin inhibitors, Myosin activator
IntroductionCytoskeletal motors have been intensively studied over the past
25 years, but our understanding of how force is produced by the
motors is still incomplete. The first crystal structure of a kinesin
motor domain (Kull et al., 1996) revealed an unexpected
structural homology between the kinesins and myosins – the
motor domain of both proteins is formed by the same core
structural elements, organized in the same way to form the
nucleotide- or filament-binding site on opposite sides of the
motor domain. These structural elements and their organization
are conserved within the kinesin and myosin superfamilies,
implying a common mechanism of force generation by the
motors. By contrast, the dyneins deviate in overall structure
from the kinesins and myosins, and presumably also in their
mechanism of energy transduction (Carter et al., 2011; Kon
et al., 2011; Kon et al., 2012; Höök and Vallee, 2012; Schmidt
et al., 2012). The focus of this Commentary is on the kinesins
and myosins, for which more is known regarding the motor
force-producing mechanism than for the dyneins. We discuss
the mechanochemical cycles of the motors and the
conformational changes they undergo, based on crystal
structures of the motors in different nucleotide states. We
propose possible force-producing mechanisms of the motors
and compare their working strokes. We also discuss small-
molecule inhibitors of the kinesins and an activator of myosin,
whose analysis has resulted in further insights into motor
function. Further information on kinesin inhibitors can be found
in a recent review (Good et al., 2011).
Motor mechanochemical cycles and forceproductionMolecular motor proteins are fascinating enzymes as they have
the ability to link chemical catalysis to the production of directed
force along a protein filament. The mechanism of force
production by motor proteins is not certain, but is thought to
involve structural changes in a deformable element of the motor
that undergoes changes in structure under load, creating strain,
followed by a strain-relieving structural change that causes the
element to recoil back into its original conformation, producing
force (Howard, 2001).
In order to produce force, motor proteins couple a chemical
cycle of ATP hydrolysis to a mechanical cycle of motor
interactions with its filament (Bustamante et al., 2004) (Fig. 1).
When coupled, the mechanochemical cycle of motor proteins can
be incredibly complex. Even at the simplest level, a minimal
chemical cycle involves ATP binding, hydrolysis, and subsequent
release of Pi and ADP. These changes occur within the relatively
small motor domain of the protein and appear to involve small
movements by specific structural elements. The mechanical cycle
is coupled to the chemical cycle and involves binding to its
filament by the motor, a lever-like movement of a rigid structural
element and/or generation of strain that produces the large
displacements observed for the motors, release of the motor from
its filament and repositioning of the force-amplifying element(s)
in preparation for the next step (Fig. 2). A key goal in the field is
to determine how these mechanochemical cycles are coupled in
different motor proteins, which amino acids and structural motifs
Commentary 1
JCS Advance Online Article. Posted on 13 March 2013
mailto:[email protected]
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are crucial for the mechanochemistry of the motors, and how
motor proteins differ to fulfil specific cellular functions.
Several key components and capabilities are common to all
motor proteins. First, motors must be able to bind to and
hydrolyze nucleotide, and then release Pi and ADP. Second, the
motor domain must be able to sense the presence or absence of c-phosphate in the nucleotide-binding pocket. Response to this
seemingly small difference, for example, when ATP rather than
ADP is bound, triggers an initial conformational response that is
then transmitted to other regions of the motor protein, inducing a
force-producing conformational change and altering interactions
between the motor and its filament. Finally, for some motors,
such as many dimeric kinesins, processive movement – the
ability to take successive steps along its filament – requires
communication between the two heads, which is thought to be
achieved by chemical and/or physical ‘gating’, in which the
attached head remains bound until a chemical or mechanical
signal, such as binding of ATP by the front head or detachment of
the rear head from the microtubule (Rosenfeld et al., 2003; Yildiz
et al., 2008; Clancy et al., 2011), causes it to release. This would
keep the two heads working synchronously, so that both heads do
not release from the filament at the same time, which would
cause the motor to diffuse rapidly away from its filament.
Structural elements involved in force productionAn important goal of the motors field is to identify the structural
elements that undergo conformational changes and the steps of
the ATP hydrolysis cycle in which they occur. Many of the
elements that are thought to play crucial roles in the
mechanochemical cycle of the kinesins and myosins have now
been identified (Box 1) through experimental approaches that
include structural analysis by X-ray crystallography and high-
resolution cryoelecton microscopy, coupled to functional studies
by mutant analysis, together with kinetic assays and cell
biological studies.
In kinesins and myosins, several conserved structural motifs in
the nucleotide-binding pocket play a crucial role. These include the
P-loop, which interacts with the nucleotide and associated Mg2+
ion, primarily through the a-, b- and c-phosphates, and two loops,switch I and switch II, which act as c-phosphate sensors (Vale,1996) (Box 1). The P-loop, also called a Walker A motif (Walker
et al., 1982), has the consensus sequence GxxxxGK(T/S), and is
one of the most common protein motifs. In kinesins and myosins,
the P-loop has a more highly conserved sequence GQ(T/
S)xSGK(T/S). In addition to the P-loop, two other motifs are
essential for motor protein function, the so-called switch I and
switch II motifs (Sablin et al., 1996) (Box 1). As these flexible
switch regions are responsible for sensing the presence or absence
of c-phosphate, they move in and out of the nucleotide-bindingpocket by several angstroms during the catalytic cycle. Their
relatively small movements are transmitted to other regions of the
motor domain and amplified in different kinesins by the neck linker
(Rice et al., 1999) or coiled-coil stalk (Yun et al., 2003), or in
myosins by the converter (Houdusse and Cohen, 1996) and lever
arm (Rayment et al., 1993) (supplementary material Movies 1, 2
and 3, respectively).
Force-producing structural pathways in kinesinsMovements in each of the two switch regions are linked through
distinct pathways of conformational change to other regions in the
Force
M.ATP
PiADP M.ADP
M.ADP.Pi
ATP
Myosin mechanochemical cycle
Mechanical cycle
Force
Chemical cycle
M
M.ATPM.ADP
M.ADP.Pi
ADP
Pi
ATP
M.ADP.Pi
M.ADP
M
Force
M.ATP
Kinesin mechanochemical cycle
M.ADP.Pi
Pi
M.ADP
ADP ATP
A
B C
Fig. 1. Motor mechanochemical cycles.
(A) Mechanical (left) and chemical (right) cycles
of a simple ATP-fueled molecular motor protein;
coupling of the mechanical and chemical cycles
produces the motor force-generating cycle.
(B) Myosin and (C) kinesin mechanochemical
cycles, simplified here to show the shift in the
cycles between the two motors. The myosin
force-producing step occurs with Pi release,
whereas ATP binding is thought to be the force-
producing step for kinesin motors (Rice et al.,
1999; Endres et al., 2006; Hallen et al., 2011).
Journal of Cell Science 126 (0)2
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motor domain. Following the switch II motif is a loop, L11,
frequently disordered in kinesins, which leads to an a-helix, helix a4(referred to as the relay helix in myosins) (Box 1), on the opposite
side of the motor domain as the P-loop and switch I. In kinesins, this
helix is a major component of the microtubule-binding interface
(Fig. 2) and has been observed in a number of different nucleotide-dependent orientations and lengths. This pathway links the
nucleotide- and filament-binding sites, allowing interactions with
+
+
+
A Kinesin-5 Eg5
B Kinesin-14 Ncd
C Myosin II
Stalk
Lever arm
Neck linker
α4-L12-α5
α6
α4-L12-α5
β-sheet
Neck linker
Neck linker
Relay helix
SH1
Converter
Myosin–ATP Myosin–no-nucleotide
ADP
Ncd–ADP Ncd–ATP
Eg5–ADP
β-sheet
Eg5–ATP
ADP
ATP
ATP
ADP
Pi
Fig. 2. Structural changes of the motors during their mechanochemical cycles. Changes in motor structure during their cycles are illustrated using crystal
structures that show one head of the dimeric motors docked onto a schematically represented filament. (A) Kinesin-5 Eg5 in the ADP state (left, PDB 1II6), the
ATP-like state (right, PDB 3HQD), and an intermediate interpolated between the two states (see supplementary material Movie 1). The microtubule-binding
elements (a4-L12-a5, magenta) interact with the microtubule. Loop L11, adjacent to helix a4, is also part of the major microtubule-binding complex, but is
frequently disordered in crystal structures, and is not shown here with the rest of the complex. Release of ADP and binding of ATP by the motor are associated
with movement of switch I (red) and a large change in the angle of the neck linker (blue), which extends toward the minus end in the ADP state (left), then swings
toward the plus end (black arrow) and docks onto the motor in the ATP-like state (right) (supplementary material Movie 1). Central b-sheet, tan; N-terminus,
yellow; helix a6 C-terminus, blue. (B) Kinesin-14 Ncd is a dimer with two heads; in stalk-rotated Ncd crystal structures, one of the heads (PDB 3L1C, chain A) is
in the same conformation as motor-ADP crystal structures – the pre-stroke state or ADP state (left) – whereas the other head (chain B) assumes a distinctly
different stalk-rotated conformation, which is thought to represent the post-stroke (Lakkaraju and Hwang, 2011) or ATP-bound state (Heuston et al., 2010) (right).
The Ncd stalk (green) tilts towards the plus end in the ADP state (left) and rotates toward the minus end (black arrow) when the motor releases ADP and binds
ATP (Endres et al., 2006; Hallen et al., 2011) (right) (supplementary material Movie 2). The microtubule-binding region conformation resembles that of kinesin-5
Eg5. (C) Myosin II in an ATP transition state (PDB 1DFL) (left) shows the lever arm (cyan) tilted towards the actin minus end; the motor undergoes large changes
as it hydrolyzes ATP and releases Pi and ADP, transitioning into the nucleotide-free state (PDB 1DFK) (right), accompanied by a large rotation of the lever arm
(black arrow) towards the plus end (right) (supplementary material Movie 3). The so-called ‘relay’ helix (magenta) corresponds to helix a4 of the kinesins; it is
kinked in the ATP-like state (left) but straightens and rotates with the lever arm, converter (orange) and SH1 helix (blue) upon Pi release and transition into the
nucleotide-free state (right). Microtubule (A), (B) and actin filament (C) schematic diagrams are oriented with the minus end to the left. Intermediate states
between crystal structures were interpolated using Chimera (Pettersen et al., 2004).
Force generation by kinesins and myosins 3
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Bo
x1.
Mo
tor
ele
men
tsin
vo
lved
info
rce
pro
du
cti
on
Kin
esi
nand
myo
sin
switc
hIa
nd
IIre
gio
ns
share
stru
ctura
land
funct
ionals
imila
ritie
sw
ithG
-pro
tein
s.
Sw
itch
I(r
ed),
afle
xible
loop
with
the
conse
rved
sequence
Nxx
SS
R,
ass
um
es
diff
ere
nt
con-
form
atio
ns
during
the
forc
e-g
enera
ting
cycl
e,
switc
hin
gbetw
een
open
(left)
and
close
dco
n-
form
atio
ns
(rig
ht)
,as
show
nin
kinesi
n-5
Eg5,
allo
win
gth
em
oto
rsto
funct
ion
asc-
phosp
hate
senso
rs.S
peci
ficin
tera
ctio
ns
ofe
ach
switc
hele
mentw
ithth
enucl
eotid
edete
rmin
ew
heth
erit
isin
an
open
orcl
ose
dco
nfo
rmatio
n(G
eeve
sand
Holm
es,
1999),
alth
ough
som
ediff
ere
nce
sexi
stbetw
een
myo
sins
and
kinesi
ns
inth
esw
itch
Iopen
confo
rmatio
n.In
the
close
dco
nfo
rmatio
n,th
esi
de
chain
hyd
roxy
lof
the
first
serine
inte
ract
sw
ithth
ec-
phosp
hate
,and
the
side
chain
of
the
seco
nd
serine
form
sa
bond
toth
ebound
Mg
2+,
movi
ng
tow
ard
sth
enucl
eotid
efr
om
itsposi
tion
inth
eopen
confo
rmatio
n(K
ull
and
Endow
,2002).
Inki
nesi
ns,
the
move
ment
of
switc
hI
from
open
tocl
ose
d
occ
urs
with
ach
ange
inits
stru
cture
from
ash
orta-h
elix
(left)
toan
ext
ended
hairpin
loop
(rig
ht)
.In
switc
hII,
alo
op
with
the
conse
rved
sequence
Dxx
GxE
,th
ecl
ose
dco
nfo
rmatio
nis
defin
ed
by
the
form
atio
nofa
hyd
rogen
bond
betw
een
the
gly
cine
andc-
phosp
hate
oft
he
bound
nucl
eotid
e(K
ull
and
Endow
,2002).
This
inte
ract
ion
posi
tions
the
loop
1–4
Åcl
ose
rto
the
nucl
eotid
eth
an
itsm
ore
variable
open
posi
tions.
When
both
switc
hre
gio
ns
are
close
d,a
cata
lytic
ally
act
ive
Pitu
be
isfo
rmed
(Kik
kaw
aand
Hiroka
wa,
2006;
Sin
dela
rand
Dow
nin
g,
2010)
(Fig
.3),
inw
hic
htw
oke
yw
ate
r
mole
cule
sin
the
act
ive
site
are
posi
tioned
toallo
wa
nucl
eophili
cattack
on
thec-
phosp
hate
by
one
of
them
,le
adin
gto
nucl
eotid
ehyd
roly
sis
(Fis
her
etal.,
1995;P
ark
eetal.,
2010).
The
nucl
eotid
e-b
indin
gP
-loop
(gre
en)
show
s
the
side
chain
sin
tera
ctin
gw
ithth
ebound
nucl
eotid
e(A
MP
?PN
P,
ora
nge).
Tw
ow
ate
r
mole
cule
s(r
ed
sphere
s)co
ntr
ibute
toth
e
oct
ahedra
lco
ord
inatio
nof
the
Mg
2+
(magenta
).
The
P-loop
confo
rmatio
ndoes
not
change
subst
antia
llyw
ithor
with
out
bound
nucl
eotid
e.
The
switc
hII
loop
(cya
n)
act
sas
ase
condary
c-
phosp
hate
senso
rby
form
ing
abond
betw
een
the
main
chain
am
ide
of
the
conse
rved
gly
cine
andc-
phosp
hate
of
the
bound
nucl
eotid
ein
the
‘clo
sed’
confo
rmatio
n;
the
gly
cine
has
move
d
back
too
far
tofo
rmth
isin
tera
ctio
nin
the
‘open’
confo
rmatio
n.
Ess
entia
lstr
uctu
ral
ele
ments
of
kin
esin
sand
myosin
sshow
nin
kin
esin
-5E
g5.
Left
,nucle
otide-b
indin
gP
-loop
(gre
en),
bound
nucle
otide
(AM
P?P
NP
,ora
nge),
sw
itch
I(r
ed)
and
sw
itch
II(c
yan).
Rig
ht,
mic
rotu
bule
-bin
din
ga4-L
12-a
5
(magenta
),a6
C-t
erm
inus
and
neck
linker
(blu
e)
and
moto
rN
-term
inus
(yello
w).
Left
,E
g5–A
DP
helic
esa4
anda5
(magenta
)blo
ck
neck
linker
dockin
g.
Rig
ht,
Eg5–A
MP
?PN
Pa4
anda5
have
rota
ted,
allo
win
gth
eneck
linker
(blu
e;
alo
ng
witha6
C-t
erm
inus)
todock
onto
the
moto
r.T
he
loop
exte
ndin
gfr
om
the
N-
term
inalb-s
trand
of
the
moto
r(y
ello
w),
the
cover
str
and,
has
not
been
fully
vis
ualiz
ed
incry
sta
lstr
uctu
res,
but
isth
ought
toin
tera
ctw
ith
the
kin
esin
-1neck
linker,
form
ing
acove
rneck
bundle
.
Fron
tB
ack
Journal of Cell Science 126 (0)4
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the nucleotide in the catalytic pocket to be transmitted to the
microtubule-binding interface, and vice-versa.
The pathway of conformational changes leading from theswitch I region is less clear, but has come into focus recentlywhen a crystal structure of kinesin 5 (Eg5, also known as KSP),
in which both switch I and switch II are closed, was solved (Parkeet al., 2010). In addition to the helix-to-loop transition of switch Ibetween the open and closed states (Box 1), comparison of the
ADP and ATP-like (bound to the nonhydrolysable ATP analogue,AMP?PNP) structures shows substantial movement of helix a3,which is adjacent to helix a2 following the P-loop. As helix a3 inkinesins is adjacent to a region containing loop L8, which alsobinds microtubules, it is possible that movements in switch Iprovide a second, distinct pathway of communication betweenthe nucleotide-binding site and the microtubule-binding interface,
allowing for fine-tuning of the kinesin mechanochemical cycle.
It is also essential that changes in the microtubule-bindingregion are transmitted to the regions of the motor that are
responsible for force generation. Interestingly, in kinesins thisappears to be governed primarily by the same movements in helixa4, discussed above, that affect interactions with the microtubuleand are observed in the ATP-bound state. The C-terminus of
helix a4 in kinesins is close to helix a6, the last helix of theconserved motor domain, as well as the N-terminus of the first b-strand of the motor domain. In kinesin family members with an
N-terminal motor domain, helix a6 is followed by the neck linker(Kozielski et al., 1997) (Box 1), which has been shown to becrucial for movement (Clancy et al., 2011), whereas in C-
terminal kinesin motors, the neck helix precedes the first b-strandof the motor domain, b-strand 1. For both N-terminal and C-terminal motors, movement of helix a4 allows the loop regionfollowing helix a6 to pack down into a small pocket on the motorcore, which is also associated with rearrangement of the end of b-strand 1. In this way, conformational changes in the nucleotide-binding region are transmitted to the C-terminal end of helix a4,resulting in nearly identical rearrangements of the loops C-terminal to helix a6 and N-terminal to b-strand 1 in both the N-and C-terminal kinesin motors (Heuston et al., 2010). At this
point, the N- and C-terminal kinesins diverge. In N-terminal,plus-end directed kinesins, these movements result in a packingof the neck linker against the motor core in the microtubule plus
direction (Box 1 and Fig. 2). In C-terminal, minus-end kinesins,the initial movements appear to trigger a rotation of the helicalneck and coiled-coil stalk in the minus-end direction (Fig. 2).
Similarities in mechanochemistry betweenkinesins and myosinsAlthough kinesin and myosin motor proteins are similar in thatthey are both powered by ATP hydrolysis, it was unexpected
when the first kinesin crystal structure was shown to overlap withthe myosin motor core structure (Kull et al., 1996). Despite analmost complete lack of sequence identity, a substantial
difference in size, and interactions with different cytoskeletonfilament tracks, the kinesin and myosin motor domains share acommon core composed of a seven-stranded b-sheet flanked bysix a-helices, three on each side of the b-sheet. Although eachfamily of motors has distinct insertions between these structuralelements, their topological order is the same, suggesting a
common evolutionary ancestor (Kull et al., 1998). Comparison ofthe kinesin and myosin catalytic pockets shows that these motorsalso share a number of common mechanistic features. Both have
P-loops as well as switch I and switch II motifs that are conservedin sequence and structure between the two motor families
(described above), and both have a helix known as the relay helixin the myosins and helix a4 in the kinesins (Fig. 2). All of theconserved active site residues make similar interactions with thenucleotide and bound Mg2+. Furthermore, comparison of myosin
and kinesin crystal structures determined in the presence ofdifferent nucleotides show similar movements andrearrangements of switch I and switch II as they transition
between open and closed conformations. This similarity in activesite elements is shared with G-proteins, which bind to andhydrolyze GTP and function as molecular switches, cycling
between GTP-bound active forms and GDP-bound inactive forms(Bourne et al., 1991). Although the P-loop, and switch I and IIregions of G-proteins share a very similar structure to those ofkinesin and myosin, their sequence motifs differ somewhat (Vale,
1996).
Even though substantial differences exist between the kinesinand myosin motors in their mechanochemistry, more similarities
than differences exist when they are closely compared. That is,kinesin and myosin hydrolyze ATP at different points in theirmechanical cycles – kinesin, while bound to microtubules, and
myosin, while detached from actin. However, this is not due tosubstantial changes in the nucleotide state-inducedconformational changes in the motor, but is caused by themechanical and chemical cycles of kinesin and myosin being out
of phase with respect to one another (Fig. 1B,C). For kinesins,microtubule binding results in loss of ADP from the motordomain, which is followed by ATP binding and hydrolysis,
coupled to a force-generating conformational change, andsubsequent release of Pi by the motor and release of the ADP-bound motor domain from the microtubule. By contrast, myosin
binding to actin induces a conformational change leading to therelease of Pi and a force-producing rotation of the lever arm.ADP is then lost, resulting in the rigor state, and ATP binding
then releases myosin from actin, at which point hydrolysisoccurs, repositioning the lever arm for the next cycle. In bothmotors, the order of conformational changes is the same, and thechanges themselves are very similar. What is different is the
function of the filament in each motor – for kinesins,microtubules act both as a nucleotide exchange factor, and,perhaps more importantly, as an activator of the motor ATPase
(Kikkawa and Hirokawa, 2006), whereas for myosin, actinfunctions exclusively as a nucleotide exchange factor.
From this common mechanochemistry, kinesins and myosins
have subtly diverged in parts of the cycle to adapt to their specificcellular roles. For example, in kinesins, the loop between switchII and helix a4 (the relay helix of myosins), loop L11, is longerthan the analogous loop in myosin and is frequently disordered in
kinesin crystal structures. It has been suggested that this loopbecomes stabilized upon microtubule binding and forms anextension of helix a4 (Hirose et al., 2006; Kikkawa andHirokawa, 2006; Nitta et al., 2008; Sindelar and Downing,2010). Helix a4 forms the primary microtubule-binding site, andacts as a fixed fulcrum upon which the plus-end directed kinesin-
1 motor domain can tilt (Sindelar and Downing, 2010). In theADP and nucleotide-free states, switch II would be open and thekinesin-1 motor tilted toward the minus direction. ATP binding
then induces a tilt towards the plus end, closing switch II andactivating the ATPase in a microtubule-dependent manner. Inmyosin, the corresponding loop between switch II and the relay
Force generation by kinesins and myosins 5
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helix is much shorter, so that the connection between them is
stronger – because ATP hydrolysis occurs when myosin is
detached from actin and functions to reposition the relay helix,
converter domain and lever arm, a tight link between the relay
helix and switch II is necessary.
One structural feature of myosin that has not yet been captured
in kinesin crystal structures is seen in the nucleotide-free state of
myosin V (Coureux et al., 2003) and myosin II (Reubold et al.,
2003). In these structures, a substantial rearrangement of the core
b-sheet has occurred, resulting in a more pronounced twistcompared with the nucleotide-bound structures (supplementary
material Movie 4). This twist causes the actin-binding cleft in
myosin to close, which is predicted to occur during rigor binding
to actin, and also moves switch I almost 10 Å away from the
nucleotide-binding site, thereby disrupting the interactions
between switch I and the nucleotide and Mg2+.
Structural analysis of the nucleotide-free conformation of
myosin and comparisons with crystal and EM structures of
kinesin suggest possible mechanisms for two steps in the motor
mechanism that remain unclear: microtubule-induced ADP
release in kinesins and actin-induced Pi release in myosin. In
kinesin, it is clear that ADP can remain bound with relatively
high affinity, even when switch I is open, as this has been
observed in a number of crystal structures. Therefore, release of
ADP upon microtubule binding could occur in kinesin if filament
binding induces a twisted-sheet conformation, thus opening
switch II even more and disrupting all interactions with the
Mg2+?ADP, as in the nucleotide-free myosin structures.
Comparison of high-resolution (10–12 Å) cryo-electron
microscopy images of kinesin–microtubule complexes in the
nucleotide-free and ADP states shows density in the b-sheetregion that is unaccounted for, and which might be due to such a
twist of the central b-sheet (Hirose et al., 2006).Following hydrolysis, it is clear that Pi must exit the
nucleotide-binding pocket by a path that differs from that of
ATP entry, as Mg2+?ADP, switch I and switch II completelycover the Pi in both kinesin and myosin. It has been suggested
that Pi release in myosin occurs through a ‘back door’ opening at
the back of the active site, which is observed in a number of
myosin structures (Yount et al., 1995; Sweeney and Houdusse,
2010; Llinas et al., 2012). However, this opening is not observed
in the rigor-like myosin structures. Furthermore, there is no
evidence for a back door in kinesin or G-proteins, and several
studies suggest that Pi could not be released through this route
(Lawson et al., 2004; Kaliman et al., 2009), casting doubt on this
theory. An alternate route for Pi release that would be consistent
for both kinesin and myosin structures involves the opening of
switch I. This could occur in a manner similar to that observed in
the nucleotide-free myosin structures, where closing of the actin-
binding cleft causes switch I to move away from the nucleotide-
binding site, or as observed in the switch I open conformations of
kinesin (Fig. 3). In either case, switch I rearrangements would
not only disrupt coordination of the Pi, but would also open up an
exit route. It should be noted that ADP would remain bound
because of interactions with the P-loop and switch II, as observed
in many kinesin crystal structures, and Pi release via the switch I
A
Eg5 SwI closed
Eg5 SwI open
MyoV SwI closed
MyoV SwI open
P-loop
P-loop
P-loop
P-loop
SwI
SwI
SwI
SwI
SwII
SwII
α4
α4
B
Fig. 3. Formation of a tube by switch I for ATP hydrolysis and Pi release. The switch I and II regions of the kinesins and myosins undergo structural changes
prior to ATP hydrolysis that result in a closed, hydrolysis-competent state. Switch I (SwI) in the closed conformation encloses the c-phosphate of the bound
nucleotide, forming a ‘Pi tube’ in the motors (Kikkawa and Hirokawa, 2006; Sindelar and Downing, 2010) (top). Following ATP hydrolysis, switch I undergoes
movements that disrupt the Pi tube and open the active site, providing a pathway for release of Pi (bottom). This pathway differs from the ‘back door’ opening in
the active site that has been proposed to provide an exit route for Pi release (Yount et al., 1995; Sweeney and Houdusse, 2010; Llinas et al., 2012),
but is now considered unlikely (Lawson et al., 2004; Kaliman et al., 2009). Formation of a Pi tube by switch I appears to facilitate ATP hydrolysis and also helps
explain the essential role of switch I in the catalytic cycle. The structures were aligned by the P-loops; bound ATP-like nucleotide (AMP?PNP for Eg5 and
ADP?BeFx for Myo V) is in the same position for each to highlight where the c-phosphate would be in the open structure. Top, SwI (red) closed; bottom, SwI
open; P-loop (green); SwII (cyan); helix a4 N-terminus (magenta). (A) Kinesin-5 Eg5 with bound AMP?PNP (orange; c-phosphate, arrow) (PDB 3HQD, top; PDB
1II6, bottom). (B) Myosin V with bound ADP (white; position of c-phosphate, arrow) (PDB 1W7J, top; PDB 1W8J, bottom). Images were rendered in PyMol
(DeLano, 2002). Myo, myosin; SwII, switch II.
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opening would not necessarily be directly coupled to release of
ADP.
Finally, it is interesting to note that the similarities betweenkinesins and myosins extend beyond the realm of motor domainsthat are related by divergent evolution, as it appears that kinesin
and myosin subfamilies have used similar approaches to solveproblems associated with being nonprocessive or processive. Ithas been known for many years that conventional myosin II
motors, such as the myosin powering muscle contraction,produce force by rotation of a rigid lever arm (Fig. 2,supplementary material Movie 3). In the case of myosin II, the
lever arm extends out of the converter domain, which is, in turn,involved in tight hydrophobic contacts with the helical lever arm.As described above, movement of switch II is linked to amovement of the relay helix, which leads to a rigid body
movement of the converter domain and lever arm, resulting in theforce-generating power stroke of myosin. Similarly, in thekinesin-14 motor Ncd, movement of helix a4 appears to lead toa large rotation of the Ncd helical neck and stalk, very similar tothe myosin power stroke (Fig. 2, supplementary material Movie2). Interestingly, both myosin II and Ncd are nonprocessive
motors; however, myosin V, a processive motor, is thought tomove along actin utilizing an extended lever arm with amechanism similar to that of myosin II. By contrast,
conventional dimeric kinesin-1 motors move processively(Howard et al., 1989; Block et al., 1990), taking multiple stepsalong a microtubule protofilament (Ray et al., 1993; Schaap et al.,2011) utilizing a different mechanism. This hand-over-hand
movement is achieved by the sequential docking and undockingof a flexible ‘neck linker’ that connects the motor domain to thecoiled-coil stalk (Kozielski et al., 1997; Rice et al., 1999) (Fig. 2,
supplementary material Movie 1). The neck linker extends inlength to allow both heads of the dimeric motor to bindsimultaneously to the microtubule and produce force, acting
together with the ‘cover strand’ (Hwang et al., 2008; Khalil et al.,2008) (Box 1). It has been suggested that strain between theheads, transmitted though the neck linkers, coordinates the
mechanochemical cycles of the two heads (see Clancy et al.,2011). Recent studies on myosin VI, a dimeric processive myosinmotor, point to a similar mechanism involving the unfolding ofan insertion between the converter domain and lever arm.
Regions of compliance in the lever arm and insertion regions thatare unique to myosin VI allow the motor to walk processively ina manner similar to that of the kinesin ‘neck-linker’ mechanism
(Ménétrey et al., 2012) with the two heads stepping along actin-binding sites and taking highly variable steps averaging 30–36 nm and up to 65 Å apart (Rock et al., 2001). It is therefore
doubly remarkable that kinesin and myosin motor proteinsevolved divergently from a common ancestor, but then appear tohave convergently evolved a similar set of strategies that areemployed in various ways to achieve processive versus non-
processive movement along their respective filaments.
Inhibitors and activators of themechanochemical cycle – insights into motorfunctionKinesin inhibitors
An early realization in the motors field was that the kinesins,
because of their essential roles in mitosis, might serve aseffective targets for drugs against cancer (Mayer et al., 1999).Small molecules that bind to specific kinesin proteins could
disrupt motor function and block cell division during tumorformation or metastasis. These compounds offer potential
advantages over currently used antimitotic drugs, many ofwhich target microtubules, in that they are expected to bespecific to dividing cells, rather than affecting all cells – theycould thus reduce the side effects caused by known microtubule
drugs due to the disruption of other microtubule-based processes.Compounds specific for given kinesin motors are also potentiallyuseful in unraveling the motor force-producing mechanism in
live cells.
The first small-molecule inhibitor to be reported that targeted akinesin was monastrol, a compound discovered in a chemical
genetics screen for inhibitors of mitosis (Mayer et al., 1999). Ascreen for cell-permeable compounds that affected mitosisidentified five that were found to have effects on mitosis butnot on microtubules. Their effects thus differed from taxol, a
widely used anti-cancer drug that affects microtubules in all cells,including those in mitosis. Monastrol, one of the five compounds,was especially interesting because of its striking effects on
dividing cells – the cells were arrested in mitosis with a ring-likearray of mitotic chromosomes attached to a monoastral spindle(Mayer et al., 1999). These cellular effects are remarkably similar
to the mutant phenotype of the kinesin-5 BimC protein (Enos andMorris, 1990) and led the authors to test the effects of monastrolon kinesin motors. Tests of monastrol on the kinesin-5 Eg5
vertebrate homologue KSP showed that the compoundspecifically blocks kinesin-5 motility in vitro, but does notinhibit kinesin-1 motility (Mayer et al., 1999). The specificity ofmonastrol for vertebrate kinesin-5 motors has been further
demonstrated by others (DeBonis et al., 2003).
Kinetic studies showed that monastrol binds to the kinesin-5motor domain and inhibits its ATPase activity but does not
compete with nucleotide or microtubule binding (Maliga et al.,2002; DeBonis et al., 2003). The effects of monastrol on kinesin-5 became clearer with the report of a crystal structure of human
kinesin-5 Eg5 complexed with monastrol (PDB 1Q0B) thatrevealed the compound bound to a newly formed site near thenucleotide-binding cleft (Yan et al., 2004) (Fig. 4). This‘induced-fit’ pocket is formed by the restructuring of loop L5
just below the active site. Remarkably, the new conformation ofL5 resembles the loop in the ATP-like state, as noted insuperpositions (Fig. 4). Thus, binding by monastrol restructures
loop L5, stabilizing the loop in the ATP-like state. Although itcan still be referred to as induced fit, it does not involve theformation of a new fold in the motor. Binding by monastrol also
induces changes in the motor distal to the binding site, includinga loop-to-helix transition in switch I, tilting of switch II helicesa4 and a5, and docking of the neck linker against the motor(Fig. 4). These latter changes in switch II and the neck linker arealso observed in kinesin motors bound to ATP analogues(Kikkawa et al., 2001; Parke et al., 2010), and are thought tobe characteristic of the ATP state of the kinesins. The two
available crystal structures (PDB 1Q0B and 1X88) both showEg5 bound to monastrol and ADP, trapped in a conformation thatresembles an ATP-like, force-producing state. Because of its
effects in slowing ATP hydrolysis, monastrol inhibits motility,reducing crosslinking and sliding of spindle microtubules individing cells.
The discovery of monastrol was a game-changer forantimitotic cancer therapeutics – it shifted the target of newscreens from microtubules to specific kinesin motors. With the
Force generation by kinesins and myosins 7
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excitement accompanying the discovery of a small-molecule
inhibitor specific for kinesin-5 came the realization that the
inhibitory activity of monastrol was too weak for it to be effective
clinically, although it was still a potentially important reagent for
use in clarifying the role of kinesin-5 in the spindle (Kapoor et al.,
2000). This recognition led to the search for more potent second-
generation kinesin inhibitors, and their discovery and
characterization. One of the most promising of the new small-
molecule inhibitors is ispinesib, which was discovered in a screen
for inhibitors of human kinesin-5 Eg5 ATPase activity (Lad et al.,
2008). Despite its structural differences compared with
monastrol, the effects of the two compounds on Eg5 are
remarkably similar – both bind specifically to Eg5 by induced
fit (Lad et al., 2008; Zhang et al., 2008; Talapatra et al., 2012),
stabilizing L5 in an ATP-like conformation, and both inhibit
ADP release and motility (Maliga et al., 2002; Lad et al., 2008)
(Box 2). Ispinesib is more potent than monastrol and is currently
being evaluated in phase II clinical trials for its effectiveness in
improving the outcome of different cancers (see http://
clinicaltrials.gov). Current success in identifying kinesin-5
inhibitors on the basis of structures of ispinesib and related
compounds is already spurring the search for further inhibitors to
circumvent the threat of resistance, which could potentially limit
their clinical use.
Other kinesin motors have also been targeted to identify new
inhibitors. Among them is kinesin-7 CENP-E, a kinetochore
motor that is thought to silence a mitotic checkpoint protein to
permit progression into anaphase (Mao et al., 2005). Loss of
CENP-E function causes prolonged mitotic delay, during which
chromosomes are clustered at either pole of the intact bipolar
αα4
NL
NL
Eg5–ADP
Eg5–AMP.PNP
α3
L8
α5
Eg5–Monastrol
Eg5–AMP.PNP
L5
α4
Eg5–ADP
α3
L8
ADP or AMP.PNP
Monastrol
Eg5–AMP.PNP
SwI
L5
Eg5–Monastrol
Eg5–ADP
ADP or AMP.PNP
Monastrol
Eg5–Monastrol
L5
Eg5–ADP
Eg5–AMP.PNP
A
C
α3
αα3
L12
B
Fig. 4. Monastrol inhibition of kinesin-5. (A) Crystal structures of kinesin-5 Eg5–ADP–monastrol (PDB 1Q0B, 1X88) show monastrol bound to a new site near
the nucleotide-binding cleft, formed by restructuring loop L5. L5 is highly mobile without monastrol, but less flexible when monastrol is bound. Superpositions
show that L5 of Eg5–ADP–monastrol (white or green, PDB 1Q0B) resembles L5 of Eg5 bound to the ATP analogue, AMP?PNP (light or medium blue, PDB
3HQD), rather than Eg5–ADP (light or dark purple, PDB 1II6) (see enlarged view below A). (B) The structural changes in Eg5–ADP–monastrol follow a pathway
from L5 to the adjacent helix a3 and then L8 [(A) and (B), green arrow]. Along this pathway, Eg5–ADP–monastrol follows Eg5–AMP?PNP somewhat more
closely than Eg5–ADP. Loop L8 contributes to microtubule binding by the motor and is adjacent to the major microtubule-binding complex L11-a4-L12-a5, and
also assumes an ATP-like conformation. Eg5–ADP–monastrol helices a4 and a5 are tilted, resembling the helices in the ATP-like state, although helix a4 is
shorter than Eg5–AMP?PNP by three turns, which might explain the weak microtubule binding by the motor bound to monastrol, compared with Eg5 in the ATP-
like state. Tilting of a4 and a5 forms an opening that allows the neck linker (NL) to dock; the motors with a docked neck linker are interpreted to represent the
force-producing ATP state (Kikkawa et al., 2001; Parke et al., 2010). (C) Despite the resemblance of L5, L8, a4, a5 and the neck linker of Eg5–ADP–monastrol to
the ATP-like state, switch I of the motor has undergone a short loop-to-helix transition, which causes it to more closely resemble Eg5-ADP than Eg5–ATP. This is
also true of the central b-sheet, indicating that the twisting of the b-sheet that is predicted to promote ADP release by the kinesins has not taken place. This is
consistent with the effects of monastrol in inhibiting ADP release, and slowing or blocking the ATP hydrolysis cycle (Maliga et al., 2002; Cochran et al., 2005).
The structural effects of monastrol on switch I could also inhibit the formation of a switch I closed conformation (Box 1), which is thought to be essential for ATP
hydrolysis. The structural changes induced by monastrol thus cause the motor, still bound to ADP and inhibited in ATPase activity, to assume a conformation that
resembles a force-producing ATP-bound state. Superposition of the protein chains was performed using Matchmaker of Chimera (Pettersen et al., 2004) and
default parameters by designating Eg5–ADP–monastrol as the reference chain and first aligning Eg5–ADP, then Eg5–AMP?PNP. The structures were displayed
and analyzed in Chimera.
Journal of Cell Science 126 (0)8
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spindle after failing to congress to the metaphase plate (Schaar
et al., 1997; McEwen et al., 2001). A screen of an organic
compound library for inhibitors of CENP-E microtubule-
stimulated ATPase activity identified GSK923295 (Wood et al.,
2010). Tests showed that the effects of GSK923295 were highly
specific to CENP-E. Assays of GSK923295 on vertebrate
cultured cells showed delayed mitosis with failure of
metaphase chromosome alignment, similar to the effects
observed previously for loss of CENP-E function.
The site of GSK923295 binding to CENP-E has been mapped to a
site adjacent to loop L5 near the active site (Box 2). Remarkably,
this site is analogous to the site of monastrol and ispinesib binding to
kinesin-5 Eg5 and might involve restructuring of L5, as is the case
for the other two small molecules. Despite the fact that the three
compounds bind to a highly similar site on the two motors, their
kinetic effects on the motors are different, probably because of
differences in their structural effects on motor microtubule-binding
elements. Further attempts to obtain a crystal structure of CENP-E
complexed with GSK923295 (Wood et al., 2010) could reveal the
mechanism of motor inhibition by the compound; it could also
potentially provide valuable information about one of the missing
states in the kinesin cycle – the no-nucleotide, or possibly the
ADP?Pi state.The discovery of small-molecule inhibitors of the kinesin
motors have thus contributed to our knowledge of their
mechanism of function and might also play have an important
clinical role in improving the outcome for patients with tumors or
malignancies, particularly those resistant to currently used
microtubule drugs, such as taxol.
Myosin activator
Small-molecule screens have also been performed on the
myosins; however, in contrast to those performed on the
kinesins, one of the screens was designed to identify
compounds that activate rather than inhibit a specific myosin
motor. A small-molecule activator specific for cardiac myosin II
was discovered in a high-throughput screen for compounds that
activate cardiac myosin in a reconstituted sarcomere or myofibril
assay (Morgan et al., 2010). Tests of the optimized compound,
omecamtiv mecarbil, showed that it accelerates Pi release and
ATP hydrolysis by cardiac myosin in the presence of actin, but
slows Pi release and ATP hydrolysis in its absence (Malik et al.,
2011). Pi release by myosin occurs at the transition between the
Box 2. Kinesin inhibition by small molecules
Kinesin-5 inhibition by ispinesib
Like monastrol (Cochran et al., 2005), ispinesib inhibits ADP
release by kinesin-5 and slows motor binding to microtubules (Lad
et al., 2008). Crystal structures show ispinesib bound to Eg5–ADP
at the same induced-fit cleft near loop L5 as monastrol (Zhang et
al., 2008; Talapatra et al., 2012). Superpositions show that L5 and
L8, the tilted switch II helices a4 and a5, and the docked neck
linker of Eg5–ADP–ispinesib are in the ATP-like conformation,
resembling Eg5–ADP–monastrol. Thus, both monastrol and
ispinesib induce structural changes in kinesin-5 at the site of
binding that are propagated to the microtubule-binding interface,
allowing the neck linker to dock. At the same time, the switch I
helix of Eg5 bound to either monastrol or ispinesib is slightly
extended and remains in an ADP-like conformation, which might
prevent its helix-to-loop transition into the closed conformation
thought to be essential for ATP hydrolysis. The central b-sheet
shows a somewhat closer resemblance to Eg5–ADP than Eg5–
AMP?PNP, suggesting that the predicted twisting of the b-sheet is
not induced by binding to either compound, inhibiting the release
of ADP.
Kinesin-7 inhibition by GSK923295
The binding site of the kinesin-7 CENP-E inhibitor, GSK923295,
has been mapped by photo-affinity labeling and mutational
analysis to a site between helices a2 and a3, adjacent to L5,
near the nucleotide-binding cleft, a site that corresponds to the
monastrol and ispinesib binding site (Wood et al., 2010).
GSK923295 inhibits Pi production or release, consistent with a
block in ATP hydrolysis, and slows microtubule-stimulated ADP
release. CENP-E bound to GSK923295 binds tightly to
microtubules, even in the presence of ADP, which is normally
the weak binding state of the motor (Wood et al., 2010). This
differs from the effect by monastrol of causing Eg5 to bind weakly
to microtubules in the presence of ADP (Cochran et al., 2005).
Overall, GSK923295 slows or blocks ATP hydrolysis by CENP-E
and traps the motor in a tight microtubule-binding state, in contrast
to the weak microtubule-binding state induced by monastrol. The
motor might be locked in the no-nucleotide state, or possibly the
ADP?Pi state, caused by the structural effects of the compound on
the motor. Thus, GSK923295 binds by induced fit to a site
corresponding to that of monastrol, yet the two inhibitors have
different effects on the motor when bound.
Box 3. Motors with increased mechanical output
Mechanical output by a motor can be increased in the following
ways:
Increased number of strokes per unit time
N An increase in the rate of Pi release, which triggers the myosinpower stroke, is predicted to result in an increased rate of ATP
hydrolysis and the number of strokes per unit time by myosin.
This was found for cardiac myosin bound to omecamtiv
mecarbil (Malik et al., 2011). A crystal structure of myosin
complexed with omecamtiv mecarbil is not yet available, but
should shed light on the mechanism by which the compound
increases the rate of Pi release by the motor.
NAn increase in the rate of ADP release, usually the rate-limitingstep in the kinesin cycle, is expected to increase ATP hydrolysis
rates and the number of strokes/unit time for kinesin. Kinesin-14
Ncd mutants that affect a conserved residue in a loop of the
central b-sheet have recently been reported that increase ADP
release and ATPase rates by the motor, resulting in faster
microtubule gliding in motility assays and strikingly elongated
spindles in vivo (Liu et al., 2012).
Increased distance per stroke
N An increase in the length of the myosin lever arm or kinesinstalk increases the gliding velocity of the motors (Stewart et al.,
1993; Chandra et al., 1993; Uyeda et al., 1996; Yun et al., 2003;
Endres et al., 2006); this has been inferred to increase the force
produced per motor, although increased force per motor has not
been directly demonstrated by single-molecule assays.
NAn increase in the angle of lever arm or stalk rotation isexpected to increase the step size (Hallen et al., 2011; Ménétrey
et al., 2012) and the force produced per motor.
NMutants that alter the free energy of motor binding to nucleotideor its filament could increase the distance per motor stroke; such
mutants have not yet been reported.
Force generation by kinesins and myosins 9
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weak and strong actin-binding state (Fig. 1). It is thought to be
required for myosin to enter the strongly bound state, which isaccompanied by rotation of the lever arm – the force-generatingstroke of the motor (Rayment et al., 1993). The overall effect of
omecamtiv mecarbil on cardiac myosin is predicted to be anincrease in the number of myosin heads interacting with actin in astrong binding state and producing force, thus it is expected toincrease mechanical output by the motor (Box 3). The binding
site for omecamtiv mecarbil was mapped using a derivative as anaffinity label and identifying labeled cardiac myosin peptides bymass spectrometry, and was found to be near the base of the lever
arm, close to the relay helix and converter. Further study of theeffects of residue changes in this region could lead to newinformation regarding the myosin force-generating mechanism –
mutational changes that increase myosin mechanical output, suchas those reported recently for kinesin-14 Ncd (Liu et al., 2012)(Box 3), would have important implications for understanding the
motor mechanism and also for potential clinical applications.
Consistent with its proposed effect in increasing mechanicaloutput by cardiac muscle, functional studies showed thatomecamtiv mecarbil increases the contractility of rat
cardiomyocytes and improves cardiac function in dogs withinduced heart failure (Malik et al., 2011). This is noteworthy,given that it is easier to disrupt motor function than to increase it,
although ‘improved’ motors could potentially be produced in anumber of different ways (Box 3). These findings have potentialfor therapeutic intervention in humans with heart disease orfailure. Recent reports of initial clinical trials in humans show
that omecamtiv mecarbil improves cardiac function in patientswith cardiac dysfunction or failure (Teerlink et al., 2011; Clelandet al., 2011).
The properties of omecamtiv mecarbil provide a strikingconfirmation of important differences between the myosins andkinesins. For the myosins, the force-producing cycle is triggeredby Pi release, which results in tight actin binding and the power
stroke, followed by ATP binding, which releases the motor fromactin. For the kinesins, the cycle begins with ADP release, whichresults in tight microtubule binding, followed by ATP binding,
which triggers the force-producing stroke of the motor, Pi releaseand release of the motor from the microtubule.
Conclusions and PerspectivesFuture progress in understanding the kinesin and myosin force-generating mechanism is likely to come from further structuralanalysis that defines the features of the tight, no-nucleotide
microtubule-bound state of the kinesins and the weak, ADP?Piactin-bound state of the myosins. The structural changes betweenthese states compared with the ATP-bound kinesin state and therigor myosin state, respectively, are expected to provide currently
missing information regarding key conformational changes thatare involved in force production by the motors. New structuralinformation, especially for kinesins with their much smaller
motor domain, could come from high-resolution cryo-electronmicroscopy, which has currently reached resolutions of 8–10 Å(Hirose et al., 2006; Kikkawa and Hirokawa, 2006; Sindelar and
Downing, 2010). These projected studies, together with thecharacterization of mutant proteins to obtain information relevantto function, should resolve currently outstanding issues, such as
the escape route of free Pi from the motor after ATP hydrolysis,and whether the central b-sheet of kinesins distorts or twists inthe same way as in myosins, and produce a more detailed
understanding of force generation by the kinesin and myosin
motors. This information will be of vital interest for comparison
with dyneins, for which unraveling the force-producing
mechanism is at a much earlier stage. The dynein motors differ
substantially from kinesins and myosins in overall structure –
their force-generating mechanism is anticipated to show
unexpected differences that will lend further insight into energy
transduction by ATP-hydrolyzing enzymes.
AcknowledgementsWe thank Anne Houdusse and Frank Kozielski for sending preprintsprior to publication, Frank Kozielski for coordinates of a crystalstructure (PDB 4AP0) prior to publication, and Amalia Cong forassistance with Fig. 2.
FundingWork on motor proteins in our laboratories is supported by grantsfrom the National Institutes of Health [grant numbers GM097079 toF.J.K. and GM046225 to S.A.E.]; and the March of DimesFoundation [grant number NO. 1-FY07-443 to S.A.E.]. Depositedin PMC for release after 12 months.
Note added in proofWhile our Commentary was being prepared for publication, webecame aware of a report by Behnke-Parks et al. noting theresemblance of Eg5–ADP–monastrol loop L5 to the ATP-likeconformation, while switch I resembles the ADP state (Behnke-Parks et al., 2011).
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.103911/-/DC1
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